Project Summary Fungal pathogens of humans are prevalent in the environment, and commonly come into contact with hosts via dispersal of vegetative spores. Although spore germination and subsequent development are recognized as critical to the initiation of fungal-host interactions, little is known about the fungal genes that govern these events. Our goal is to leverage comparative genomics, evolutionary biology and fungal pathogenesis to define genes that are essential for spore germination, subsequent growth, and host colonization in an evolutionarily diverse group of fungi. We have chosen fungi with a range of abilities to cause disease upon interaction of spores with mammalian hosts, including primary, opportunistic, and nonpathogenic species. We will take advantage of a highly effective and innovative pipeline that reveals genes whose evolving roles have led to phenotypic differences among these species. PIs Trail and Townsend have defined a paradigm that brings together comparative genomics, developmental biology, and transcriptomics into a single, unified phylogenetic framework that will identify key genes that govern spore germination and outgrowth in these fungi. The linchpin of our approach is use of the evolutionary relationship between the fungi to infer genes whose expression has been altered during evolution from their ancestral state to each present-day lineage, thus allowing specific traits (such as pathogenesis) to evolve. In our recently published work and preliminary data, this approach was immensely powerful for identifying genes whose evolving role led to developmental and phenotypic differences among species during (1) fungal sexual development and (2) spore germination and early infection during fungal pathogenesis of plants. We will use a common medium to germinate spores from the following fungi: the primary pathogens Histoplasma capsulatum and Coccidioides posadasii, the opportunistic pathogens Aspergillus fumigatus, Fusarium oxysporum, and Chaetomium elatum, the infrequent opportunistic pathogen Aspergillus nidulans, and the non-pathogenic Neurospora crassa. We will subject these fungi to transcriptomics over a time-course of germination and subsequent development under temperature conditions relevant to germination in the environment vs. in a mammalian host. We will reconstruct evolutionary changes of gene expression across these multiple species to identify genes that have undergone recent shifts in gene expression, in particular shifts that occurred along the shared ancestral branches where key traits (such as the ability to colonize mammals) have evolved. These experiments will yield a high-confidence set of candidate genes whose function is expected to be critical for spore germination and development in each organism. We will use gene knock-out technology to interrogate the function of these candidate genes in spore germination and development. These studies will identify potential targets for diagnostic, prophylactic, and vaccine interventions for ubiquitous fungal infections of humans.